The broad goal of the proposed research is to investigate how lysine acetylation modifications of mitochondrial proteins regulate drug-induced liver injury (DILI). Acetyl modification of mitochondrial proteins is beginning to be recognized as a widespread post-translational modification, found on a diverse range of mitochondrial metabolic pathway proteins. SIRT3 is the-major mitchondiral deacetylace, and to date its deacetylation function has been shown to play a role in multiple biological insults including redox, genotoxic arid nutrient stressors. We recently found that SIRT3-/-mice are resistant to acetaminophen-induced liver injury. We hypothesize that increased acetylation of mitochondrial proteins might play an important role in DILI. We have identified several novel SIRT3 deacetylation targets, including mitochondrial aldehyde dehydrogenase 2 (ALDH2), apoptosis-inducing factor (AlF), ATP synthetase a-subunit (ATPa) and heat shock protein 10 (HSP10), which are indirectly implicated as targets in the development of DILI. Our preliminary data additionally shows a greater retention pf ALDH2 activity, and a reduction in toxic aldehyde levels, in S1RT3-/- mice. Furthermore, we show that in SIRT3-/- mice deacetylation ofALDH2 diminished the capacity of toxic acetaminophen metabolites to bind to ALDH2, and also have identified lysine residue functioning to this binding modulation. To further test our hypothesis, we will employ ALDH2 and AIF as the index proteins to explore and delineate the role that modulation of mitochondrial protein acetylation plays in DILI. In the proposed study, we will and investigate the role of lysine acetylation status in acetaminophen (APAP) metabolite binding, and its effect on ALDH2 function; susceptibility to acetaminophen hepatoxicity; and the potential therapeutic application of the ALD2 activator (Alda-1) in APAP hepatotoxicity. We will also investigate the acetylation status of AIF, ATPa, and HSP10 in regulating acetaminophen hepatotoxicity. We believe that this study will unmask novel functions of protein acetylation in modulating susceptibility to xenobiotic-induced liver injury. Increased understanding of this biology will have broad implications, aiding the development of strategies/therapies to alleviate the consequences of drug-induced liver injury.